Margarita Angerosa

Effects of angiotensin II subtype 1 receptor blockade by losartan on tubulointerstitial lesions caused by hyperoxaluria.

PURPOSE Hyperoxaluria is a recognized cause of tubulointerstitial lesions and this circumstance could contribute to cause chronic renal disease. The renin-angiotensin system has a critical role in the development of interstitial fibrosis, mostly by angiotensin II type 1 receptor stimulation of pro-fibrotic mechanisms. We evaluated whether angiotensin II type 1 receptor blockade prevents oxalate renal lesions. MATERIALS AND METHODS We divided 2-month-old male Sprague-Dawley rats into 4 groups, namely group 1-control, group 2-hyperoxaluria, group 3-hyperoxaluria plus losartan and group 4-losartan. For 4 weeks groups 2 and 3 received 1% ethylene glycol (precursor for oxalates) in drinking water. Losartan (40 mg./kg. body weight) was administered in groups 3 and 4 daily. At the end of the study renal lesions were evaluated using anti-alpha-smooth muscle actin, anti-collagen type III, anti-monocytes/macrophages and anti-transforming growth factor-beta1 antibodies. To evaluate oxidative stress in renal tissue total glutathione and thiobarbituric acid reactive substances in kidney homogenates were determined. Regarding renal functional parameters, creatinine clearance and urinary albumin excretion were also studied. RESULTS Despite similar urinary oxalate levels compared with group 2 group 3 rats showed fewer tubulointerstitial lesions, consisting of significant lower scores for tubular atrophy, unspecific inflammatory cell infiltrate, ED1 mouse anti-rat monoclonal antibody (Serotec, Ltd., Oxford, United Kingdom) (monocytes/macrophages), crystal deposits, interstitial fibrosis, alpha-smooth muscle actin, collagen type III and tubulointerstitial transforming growth factor-beta1. Moreover, urinary albumin excretion and creatinine clearance were significantly improved in group 3 (p <0.01). Higher total glutathione and lower thiobarbituric acid reactive substances were also observed in this group (p <0.01). Thiobarbituric acid reactive substances were the most important and significant independent variable correlating with interstitial fibrosis (t ratio 4.867, p <0.04). CONCLUSIONS We believe that the renal-angiotensin system interaction by losartan produces a beneficial effect against renal lesions caused by hyperoxaluria through a number of actions, including a reduction in crystal formation in the tubular fluid, inflammatory reaction control and interaction with oxidative stress. These factors lead concurrently to preserve tubular epithelial cell and renal interstitium integrity. In addition, these results suggest that the principal mechanism of action should be mediated by angiotensin II type 1 receptors.

Comparison of the renal, cardiovascular and hepatic toxicity data of original intravenous iron compounds

BACKGROUND Intravenous (i.v.) iron is essential for managing haemoglobin levels in haemodialysis patients. However, i.v. iron may cause variable degrees of toxicity. This is mainly related to the pharmacological characteristics of any given i.v. iron compound. METHODS This blinded study examines the effects of five i.v. iron preparations on haemodynamic and functional parameters. Sixty Sprague-Dawley rats (n = 10/group) received high or low molecular weight (HMW/LMW) iron dextran, ferric gluconate (FG), ferric carboxymaltose (FCM), iron sucrose (ISC) or isotonic saline solution (control). Five i.v. doses of iron (40 mg iron/kg) or saline were administered over 4 weeks. RESULTS Systolic blood pressure was significantly reduced in the LMW dextran group, whereas serum iron and percentage transferrin saturation were significantly elevated in all treatment groups. Creatinine clearance was reduced and urinary protein excretion increased in the FG group only (P < 0.01). Liver enzyme levels in the blood were increased (P < 0.01) in the FG and two dextran groups compared with the FCM and ISC groups. Analysis of liver, heart and kidney homogenates showed a significant increase in catalase and malondialdehyde levels in the FG group, and an increase in CuZn-superoxide dismutase and glutathione (GSH) peroxidase activity accompanied with a decrease in the reduced-to-oxidized GSH ratio in the FG and two dextran groups (P < 0.01). Tumour necrosis factor alpha and interleukin-6 levels were significantly elevated in liver, heart and kidney samples from the FG and two dextran groups but not the FCM, ISC or control groups. CONCLUSIONS These findings indicate that FG and HMW/LMW iron dextran have less favourable safety profiles than FCM and ISC in normal rats.

Antifibrotic effects of pioglitazone on the kidney in a rat model of type 2 diabetes mellitus.

BACKGROUND Recent evidence suggests that treatment of type 2 diabetes with thiazolidinediones [peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists], ameliorates glomerulosclerosis and tubulointerstitial fibrosis in the rat kidney. In the current work, we have investigated whether these drugs, and specifically pioglitazone (PGT), act by preventing fibrosis and kidney dysfunction mainly through antioxidant and anti-inflammatory effects, independently of glycaemic control. METHODS Male 2- to 3-month-old obese Zucker fa/fa (OZR) and ZDF fa/fa rats (ZDFR), and their control the lean Zucker rat (LZR), were used. Diabetic rats were given either a low dose (0.6 mg/kg/day) or a high dose (12 mg/ kg/day) of PGT in the chow for 2 or 4-5 months. Glycaemia, blood pressure, creatinine clearance and proteinuria were determined, and the underlying histopathology was defined with markers of fibrosis, glomerular damage, oxidative stress and inflammation by immunohistochemistry/ quantitative image analysis in tissue sections, and western blots and ad hoc assays in fresh tissue. RESULTS PGT at low doses given for 4-5 months considerably reduced blood pressure, proteinuria and creatinine clearance. This was associated with amelioration of renal tissue damage and fibrosis, evidenced by the glomerulosclerosis, tubulointerstitial fibrosis, tubular atrophy and podocyte injury indexes, and of oxidative stress and inflammation, as shown by the decrease in the respective markers, although glycaemia remained high and obesity was not affected. CONCLUSIONS These results indicate that low doses of PGT ameliorate renal fibrosis and preserve renal function in this animal model of metabolic syndrome, independently of glycaemic control or effects on body weight.

Comparison of Oxidative Stress and Inflammation Induced by Different Intravenous Iron Sucrose Similar Preparations in a Rat Model

Iron sucrose originator (ISORIG) has been used to treat iron deficiency and iron deficiency anemia for decades. Iron sucrose similars (ISSs) have recently entered the market. In this non-clinical study of non-anemic rats, five doses (40 mg iron/kg body weight) of six ISSs marketed in Asian countries, ISORIG or saline solution (control) were administered intravenously over four weeks to compare their toxicologic effects. Vasodilatory effects, impaired renal function and hepatic damage were only observed in the ISS groups. Significantly elevated serum iron and transferrin saturation levels were observed in the ISS groups suggesting a higher release of iron resulting in higher amounts of non-transferrin bound (free) iron compared to ISORIG. This might explain the elevated oxidative stress and increased levels of inflammatory markers and antioxidant enzymes in the liver, heart and kidneys of ISS-treated animals. Physico-chemical analyses showed that the molecular structure of most of the ISSs differed greatly from that of the ISORIG. These differences may be responsible for the organ damage and oxidative stress observed in the ISS groups. Significant differences were also found between different lots of a single ISS product. In contrast, polarographic analyses of three different ISORIG lots were identical, indicating that the molecular structure and thus the manufacturing process for ISORIG is highly consistent. Data from this study suggest that ISSs and ISORIG differ significantly. Therefore, before widespread use of these products it would be prudent to evaluate additional non-clinical and/or clinical data proving the safety, therapeutic equivalence and interchangeability of ISSs with ISORIG.

Differences between original intravenous iron sucrose and iron sucrose similar preparations.

Iron sucrose (Venofer; reference) has a good safety record and is prescribed in patients with anaemia and chronic kidney disease worldwide, but various iron sucrose similar (ISS) preparations are now utilized in clinical practice. This study evaluates possible differences between iron sucrose and ISS preparations on haemodynamic and oxidative stress markers in normal rats. 60 male and 60 female Sprague Dawley rats were divided into four groups and assigned to receive commercially available ISS test 1, ISS test 2, reference or isotonic saline solution (control). A single i.v. dose of iron (40 mg/kg) or saline (equivalent volume) was administered after 24 h and every 7 days for 4 weeks. Blood samples were collected for biological assessment of haemoglobin (Hb), serum iron and percentage transferrin saturation (TSAT), and urine samples were collected to investigate creatinine clearance and proteinuria. Animals were sacrificed after receiving an i.v. dose on days 1, 7 and 28, and kidney, liver, and heart homogenates were then collected to determine antioxidant enzyme levels. Tissues were processed using Prussian blue and immmunohistochemistry techniques to identify iron deposits, tissue ferritin and pro-inflammatory markers. Systolic blood pressure was significantly reduced in the ISS groups relative to the reference and control groups after 24 h and on days 7, 14 and 21 (p < 0.05). Creatinine clearance was reduced (p < 0.01) and proteinuria marked (p < 0.01) in the ISS groups at 24 h and on days 7 and 28 relative to the reference and control groups which did not differ throughout the study. Liver enzymes were also increased in the ISS groups at 24 h and on days 7 and 28. Both ISS test 1 and ISS test 2 groups presented a significant increase in catalase, thiobarbituric reactive species, Cu, Zn-superoxide dismutase (CuZnSOD) and glutathione peroxidase activity, and a decrease in glutathione levels (p < 0.01) in the liver, heart and kidney at 24 h and on day 7 relative to the reference and control groups. Serum iron and percentage TSAT were elevated in all groups (except control) (p < 0.01) but no differences in Hb concentration were observed between them. Finally, levels of the proinflammatory markers TNF-alpha and IL6 were significantly elevated in the ISS groups (liver, heart and kidney) compared with the reference and control groups on day 28 (p < 0.01). These findings suggest significant differences between the reference and ISS test 1/ISS test 2 regarding oxidative stress and the inflammatory responses of liver, heart and kidneys in normal rats. A possible explanation for these observations could be the stability of the iron complex.

Cardiovascular protective effects of nebivolol in Zucker diabetic fatty rats.

Background Although effective in reducing blood pressure, therapy with a first-generation β-blocker is currently controversial in metabolic syndrome due to its negative impact on carbohydrate and lipid metabolism. Objective and design We evaluated the effects of nebivolol, a third-generation highly selective β-blocker with additional vasodilating activity, versus the traditional β-blocker atenolol in controlling functional and morphological cardiovascular damage in a rat model of metabolic syndrome. Methods During 6 months, Zucker diabetic fatty (ZDF) rats and control lean Zucker rats (LZR) were studied. The experimental groups were: untreated ZDF, ZDF along with nebivolol, ZDF along with atenolol and LZR. Blood pressure, plasma insulin, triglycerides, cholesterol, glucose and platelet aggregation were evaluated. Malondialdehyde, reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, CuZn superoxide dismutase, catalase and glutathione peroxidase were determined in heart homogenates and transforming growth factor β1 and plasminogen activator inhibitor-1 (PAI-1) expression, by immunohistochemistry (IHC). Vascular reactivity, vascular cell adhesion molecule-1, platelet endothelial cell adhesion molecule-1, PAI-1, enhanced nitric oxide synthase and collagen expression were evaluated in aorta. Results Nebivolol and atenolol presented a similar reduction in blood pressure. However, nebivolol showed a better lipid profile, preserved left ventricular function, a significant control in left ventricular geometry and moderated left ventricular hypertrophy versus atenolol. Significant reduction in platelet aggregation and a substantial endothelium-dependent and endothelium-independent relaxation in vessels were also shown in the nebivolol group versus atenolol group. Antioxidant defenses were preserved by nebivolol with a reduction in oxidative stress parameters. Vascular cell adhesion molecule-1, platelet endothelial cell adhesion molecule-1, PAI-1 and eNOS were favorably modulated with nebivolol in vessel wall. TGFβ1, PAI-1 and accumulation of collagen-III and collagen-I were also diminished in heart with nebivolol. Conclusion The present study provides substantial information supporting an actual protective role of nebivolol in comparison with atenolol in experimental metabolic syndrome.

Enalapril Prevents Tubulointerstitial Lesions by Hyperoxaluria

Hyperoxaluria is a recognized cause of tubulointerstitial lesions, and this could contribute to development of hypertension and chronic renal failure. Enalapril has been effective against the progression of tubulointerstitial lesions in various animal models. The aim of the present study was to evaluate the usefulness of enalapril on the tubulointerstitial damage produced by oxalates. Two-month-old male Sprague-Dawley rats were separated into 4 groups, control with tap water (G1), hyperoxaluric (G2), hyperoxaluric+enalapril (G3), enalapril (G4), for 4 weeks. G2 and G3 rats were given 1% ethyleneglycol (ETG, precursor for oxalates), and G3 and G4 rats were given enalapril 20 mg/L in drinking water. At the end of the study, we evaluated renal tubulointerstitial lesions by a semiquantitative score. Urine albumin excretion, serum and urine nitric oxide production, tubulointerstitial immunostaining by alpha-smooth muscle actin, transforming growth factor-beta1, and collagen type III were measured. Rats belonging to the hyperoxaluric group treated with enalapril (G3) showed fewer tubulointerstitial lesions (1.3+/-0.2 versus 3+/-0.2; P<0.01), lower urine albumin excretion (8+/-2 mg/d versus 25+/-2 mg/d; P<0.01), less percentage of alpha-smooth muscle actin in renal interstitium (2+/-0.4% versus 13.5+/-2.4%; P<0.01), less percentage of transforming growth factor-beta1 in tubulointerstitial area (3.3+/-1% versus 13.3+/-2. 1%; P<0.01), less percentage of collagen type III interstitial deposition (0.7+/-0.5% versus 7+/-2.6%; P<0.01), and increased NO production in serum as well as urine (both P<0.01), when compared with the hyperoxaluric group not treated with enalapril (G2). Considering these data, we believe that enalapril, by several mechanisms of action, could provide an important benefit in the prevention of inflammatory response, transforming growth factor-beta1 tubulointerstitial production, collagen type III interstitial deposition, and finally, the progressive tubulointerstitial fibrosis caused by oxalates.

PROTECTIVE ROLE OF ENALAPRIL FOR CHRONIC TUBULOINTERSTITIAL LESIONS OF HYPEROXALURIA

PURPOSE Hyperoxaluria is a recognized cause of tubulointerstitial lesions and it may contribute to chronic renal failure. In previous studies we demonstrated that enalapril was effective against the progression of tubulointerstitial lesions in a 4-week hyperoxaluria rat model. We evaluated whether the action of enalapril on the tubulointerstitial lesions produced by hyperoxaluria persisted for a long period. MATERIALS AND METHODS Two-month-old male Sprague-Dawley rats were divided into 4 groups of 12 each, including 1--control animals given tap water, 2--animals with hyperoxaluria, 3--animals with hyperoxaluria plus enalapril, 4--animals with enalapril. Hyperoxaluria in groups 2 and 3 rats was induced by administering 1% ethylene glycol, a precursor for oxalates, in the tap water continuously throughout the whole study. Meanwhile, groups 3 and 4 received 20 mg./l. enalapril in the drinking water. At the end of the study renal tubulointerstitial lesions were evaluated by immunostaining using monoclonal antibodies against macrophage infiltrates (ED1), tubulointerstitial alpha-smooth muscle actin and transforming growth factor-beta1. The lesions were quantified by semiquantitative scores. Creatinine clearance and urinary albumin excretion were also determined. RESULTS There was no difference in urine oxalate excretion in groups 2 and 3. Group 3 rats treated with enalapril showed fewer tubulointerstitial lesions than nontreated group 2 rats, as indicated by the mean scores plus or minus standard error of mean for inflammatory infiltrate (2.16 +/- 0.2 versus 0.83 +/- 0.16), tubular atrophy (2 +/- 0.27 versus 0.66 +/- 0.14), interstitial fibrosis (2.5 +/- 0.15 versus 0.5 +/- 0.1), glomerular ED1 (1.75 +/- 0.25 versus 0.16 +/- 0.11), interstitial ED1 (2.33 +/- 0.18 versus 0.58 +/- 0.10) tubular transforming growth factor-beta1 (2.09 +/- 0.08 versus 0.91 +/- 0.14), interstitial transforming growth factor-beta 1 (2.33 +/- 0.22 versus 0.66 +/- 0.12), tubulointerstitial alpha-smooth muscle actin (2.91 +/- 0.22 versus 0.83 +/- 0.16), lower urinary albumin excretion (35.5 +/- 2.7 mg. daily versus 10.9 +/- 1) and higher creatinine clearance (2.29 +/- 0.04 ml. per minute versus 2.54 +/- 0.03, all p <0.05). CONCLUSIONS Based on our results we believe that enalapril would provide a beneficial effect against chronic tubulointerstitial lesions caused by oxalates.

Antifibrotic Effects of Pioglitazone at Low Doses on the Diabetic Rat Kidney Are Associated with the Improvement of Markers of Cell Turnover, Tubular and Endothelial Integrity, and Angiogenesis

Background/Aims: Pioglitazone and other thiazolidinediones are renoprotective in diabetic nephropathy at doses that normalize glycemia, presumably as a consequence of glycemic control. However, low doses of pioglitazone that did not normalize glycemia in rat models of type 2 diabetes prevented tubulointerstitial fibrosis and glomerulosclerosis through counteracting inflammation, oxidative stress, cell cycle arrest, and fibrosis. The current work tested whether this low-dose treatment also reduces other fibrosis and inflammation factors in the diabetic kidney and prevents tubular cell loss, endothelial damage, and abnormal angiogenesis. Methods: ZDF fa/fa rats (ZDF) were fed for 4 months chow with 0.001% pioglitazone, and the untreated ZDF and the non-diabetic lean Zucker rats (LZR) received regular chow. Proteinuria, creatinine clearance, blood pressure, and renal quantitative histopathology markers were determined. Results: Correction of renal function in ZDF by pioglitazone, occurring with a glycemia >250 mg/dl, was accompanied by normalization of the renal levels of connective tissue growth factor and fibronectin (fibrosis), TNF-α, interleukin-6 and MCP-1 (inflammation), megalin (tubular cells), the PCNA/caspase-3 ratio (positive cell turnover), VEGF (abnormal angiogenesis), and the ratio between eNOS and iNOS (endothelial dysfunction). Conclusion: This supports mechanisms for the renoprotective effects of pioglitazone in diabetes additional to glycemic control.

Assessment of the extent of oxidative stress induced by intravenous ferumoxytol, ferric carboxymaltose, iron sucrose and iron dextran in a nonclinical model.

Intravenous (i.v.) iron is associated with a risk of oxidative stress. The effects of ferumoxytol, a recently approved i.v. iron preparation, were compared with those of ferric carboxymaltose, low molecular weight iron dextran and iron sucrose in the liver, kidneys and heart of normal rats. In contrast to iron sucrose and ferric carboxymaltose, low molecular weight iron dextran and ferumoxytol caused renal and hepatic damage as demonstrated by proteinuria and increased liver enzyme levels. Higher levels of oxidative stress in these tissues were also indicated, by significantly higher levels of malondialdehyde, significantly increased antioxidant enzyme activities, and a significant reduction in the reduced to oxidized glutathione ratio. Inflammatory markers were also significantly higher with ferumoxytol and low molecular weight iron dextran rats than iron sucrose and ferric carboxymaltose. Polarographic analysis suggested that ferumoxytol contains a component with a more positive reduction potential, which may facilitate iron-catalyzed formation of reactive oxygen species and thus be responsible for the observed effects. Only low molecular weight iron dextran induced oxidative stress and inflammation in the heart.